The long-term goal of the proposed research is to define the anatomy and control of meiotic recombinogenic regions in mammalian genomes. Studies in a variety of models have demonstrated that recombination occurs in distinct regions coined hotspots during meiosis I. Indeed, it has been estimated that ~1-2% of the genomes of higher eukaryotes are recombinogenic, while the remainder of the genome is left in the "cold". However, very little is known regarding the cis-acting features that define recombination hotspots, nor how trans-acting factors control meiotic recombination in complex mammalian genomes. To define the anatomy and regulation of mouse recombination hotspots, in Aim 1 we will identify and characterize novel recombination hotspots in the mouse. We will focus our efforts on defining the recombination hotspots of mouse chromosome 19, and will characterize their rates and crossover/non-crossover (CO/NCO) profiles by direct sperm and oocyte typing, and by analyzing the genomic structure and plasticity of these loci in wild-house mouse populations. Our Preliminary Studies using recombinant inbred mice as a crossover library have established that we can rapidly identify bona fide recombination hotspots. The identification of recombination hotspots will allow us in Aim 2 to define the cis-acting features that direct recombination hotspot activity in the mouse. Our focus here is to interrogate the influence of local differences of cis-acting sequences at the cores of recombination hotspots. We will study nucleosome occupancy at hotspots and assess their role in influencing double strand break (DSB) initiation sites. Our preliminary data, using a refined FACS based method to purify the various meiotic stages, have provided exquisite direct insight into the nucleosome occupancy at recombination hotspots and the surrounding recombinogenic inert regions. Using the power of recombinant inbred strains we propose to assess the long-range genetic control of recombination hotspots activity depending on the interplay between the paired homolog they are faced to and their intrinsic local nature. Collectively, the proposed studies will provide fundamental knowledge regarding the anatomy and control of meiotic recombination hotspots in complex mammalian genomes, and they may also provide very important insights into how these molecular machines go awry in disease states such as infertility and cancer. PUBLIC HEALTH RELEVANCE: The proposed research of this revised R01 grant application is directly relevant to our understanding of genome turnover and evolution, where we seek to define the anatomy and control of recombination hotspots, which are preferred sites where chromosomes reshuffle between each generation. Surprisingly, these hotspots only represent a small fraction (1% to 2%) of the whole genome, and to date most analyses of these sites have been limited to the yeast, which have a much smaller and simpler genome than those of higher eukaryotes. The proposed studies will define the anatomy and activity of new recombination hotspots in the mouse genome, and will assess their control by factors known to play critical roles in recombination. These studies will lay the foundation for understanding how recombination is controlled in complex mammalian genomes.